Recombinant Yarrowia lipolytica with high lycopene production, construction method and application thereof

Abstract

The present invention provides an application of a phytoene dehydrogenase gene RccarB derived from Mucor circinelloides in increasing the yield of lycopene produced by fermentation of recombinant Yarrowia lipolytica. The present invention also provides a phytoene synthase gene mutant RscrtB D179R , which is to mutate the D179 position of the RscrtB gene from alanine to arginine. The present invention also provides RscrtB D179R Application in improving the yield of lycopene produced by fermentation of recombinant Yarrowia lipolytica. The present invention constructs a recombinant Yarrowia lipolytica with high lycopene yield, wherein the recombinant Yarrowia lipolytica expresses RscrtB D179R , RccarB and geranylgeranyl pyrophosphate synthase genes. The recombinant Yarrowia lipolytica of the present invention has a lycopene production of 1.275 g / L in shake flask fermentation and 11.89 g / L in a 5L fermenter, which is significantly ahead of the prior art.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant Yarrowia lipolytica with high lycopene production, a construction method of the recombinant Yarrowia lipolytica, and an application of the recombinant Yarrowia lipolytica in producing lycopene. Background Art

[0002] Lycopene has strong antioxidant function and is rated as a Class A nutrient by the World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO). It has the functions of preventing atherosclerosis, preventing and treating hyperlipidemia, slowing the development of cardiovascular diseases, and preventing and fighting cancer. It can be used in food, health care products and other industries.

[0003] The production methods of lycopene include natural extraction, chemical synthesis and microbial fermentation. Among them, the microbial fermentation method has the advantages of simple process and high production efficiency and has attracted attention. The main strain for industrial production of lycopene is Blakeslea trispora, but the products are mostly mixed carotenoids and the operation process is complicated; Escherichia coli and yeast engineering bacteria that produce lycopene have been successfully constructed, but Escherichia coli hides certain safety hazards, so using yeast to produce lycopene has certain advantages. Chinese patent application CN 200510010697.1 discloses the introduction of exogenous crtE, RscrtB, and crtI genes into Pichia pastoris to construct a lycopene synthesis pathway; Chinese invention patent application CN 202211059315.4 discloses the introduction of exogenous OcrtE, ORscrtB, and OcrtI genes into Pichia pastoris to construct a lycopene synthesis pathway, and strengthen NADPH synthesis, and regulate the fermentation strategy in a 5L fermenter to achieve a lycopene yield of 7.5g / L. Chinese invention patent application CN202110030418.7 discloses the construction of a lycopene synthesis pathway in brewer's yeast, increasing lycopene production by adding linoleic acid, and achieving a fermentation yield of 301 mg / L (86.04 mg / g) in a shake flask.

[0004] Yarrowia lipolytica (Y.lipolytica) is a safe oil-producing microorganism with a clear genetic background. It can provide the precursor compound geranylgeranyl pyrophosphate (GGPP) and energy supply substances acetyl COA, NADPH and ATP. Yarrowia lipolytica itself can produce geranylgeranyl pyrophosphate (GGPP), a precursor compound for lycopene synthesis, and as an oil-producing microorganism, it can provide sufficient storage space for lycopene. Yang Kaixin expressed crtE, RscrtB, crtI, HMG1, and GGS1 genes in Yarrowia lipolytica and located them in peroxisomes, knocked out the gut2 gene in the lipid synthesis pathway, and the fermentation yield in a shake flask reached 70.52 mg / L (Yang Kaixin. Heterologous synthesis of lycopene in Yarrowia lipolytica [D]. Huazhong University of Science and Technology, 2020), but the yield is still low.

[0005] Phytoene synthase is the first synthase in the carotenoid biosynthesis pathway, which catalyzes the formation of phytoene from two molecules of GGPP. A large number of studies have shown that phytoene synthase is the rate-limiting enzyme in the carotenoid metabolic pathway. Based on this, the current problem is to screen phytoene synthases from different sources, modify the phytoene synthase from the most active source, and increase the supply of precursor compounds to improve the lycopene production capacity of recombinant Yarrowia lipolytica. Summary of the invention

[0006] The purpose of the present invention is to construct a recombinant Yarrowia lipolytica with high lycopene production.

[0007] The idea of ​​the present invention is to screen the phytoene synthase genes RscrtB, PacrtB, SacrtB, RccarRP from different sources in Yarrowia lipolytica. E78K and phytoene hydrogenase genes RccarB, BtcarB, PacrtI; mutate the phytoene synthase gene from the best source and verify whether it has the effect of further increasing the yield; then further screen the geranylgeranyl pyrophosphate synthase genes XdcrtE, PacrtE, SaGGPPs, YlGGPPs; express the key precursor genes tHMGR, ERG12, IDI and ERG20 in the MVA pathway; introduce exogenous PK and PTA genes to increase the supply of acetyl-CoA, and finally construct a recombinant Yarrowia lipolytica with high lycopene production.

[0008] Based on this, the present invention provides the use of a phytoene dehydrogenase gene RccarB derived from Mucor circinelloides in increasing the yield of lycopene produced by fermentation of recombinant Yarrowia lipolytica, wherein the recombinant Yarrowia lipolytica expresses a phytoene synthase gene.

[0009] Furthermore, the present invention provides a phytoene synthase gene mutant RscrtB D179R , characterized in that the mutant is a mutant in which the D179 position of the phytoene synthase gene RscrtB from Mucor circinelloides is mutated from alanine to arginine.

[0010] The present invention also provides a phytoene synthase gene mutant RscrtB D179R Application in improving the yield of lycopene produced by fermentation of recombinant Yarrowia lipolytica, wherein the recombinant Yarrowia lipolytica expresses a phytoene dehydrogenase gene.

[0011] On the other hand, the present invention also provides the use of the geranylgeranyl pyrophosphate synthase gene in improving the yield of lycopene produced by fermentation of recombinant Yarrowia lipolytica, wherein the recombinant Yarrowia lipolytica expresses the phytoene synthase gene and the phytoene dehydrogenase gene.

[0012] Preferably, the geranylgeranyl pyrophosphate synthase gene is XdcrtE from Phaffiarhodozyma, PacrtE from Pantoea agglomerans and / or SaGGPPs from Sulfolobus acidocaldarius.

[0013] Furthermore, the present invention also provides a method for overexpressing the phytoene synthase gene mutant RscrtB. D179R , the application of the phytoene dehydrogenase gene RccarB from Mucor circinelloides and the geranylgeranyl pyrophosphate synthase gene XdcrtE from Phaffia rhodozyma in improving the production of lycopene by recombinant Yarrowia lipolytica fermentation.

[0014] The present invention also provides a recombinant Yarrowia lipolytica with high lycopene production, wherein the recombinant Yarrowia lipolytica is obtained by expressing RscrtB in the Yarrowia lipolytica polf strain. D179R , RccarB and geranylgeranyl pyrophosphate synthase genes, the RscrtB D179RThe gene is a mutant obtained by mutating the D179 position of the phytoene synthase gene RscrtB from Mucor circinelloides from alanine to arginine, the RccarB gene is a phytoene dehydrogenase gene from Mucor circinelloides, and the geranylgeranyl pyrophosphate synthase is selected from XdcrtE from Phaffia rhodozyma, PacrtE from Pantoea agglomerans and SaGGPPs from Sulfolobus acidocaldarius.

[0015] Preferably, the geranylgeranyl pyrophosphate synthase gene is the geranylgeranyl pyrophosphate synthase gene XdcrtE derived from Phaffia rhodozyma.

[0016] The present invention also provides a method for constructing the above-mentioned recombinant Yarrowia lipolytica, which comprises the following steps:

[0017] (1) Expression vector construction

[0018] (1.1) Artificial synthesis of RscrtB D179R , RccarB, XdcrtE, PacrtE and SaGGPPs genes (as shown in SEQ ID No. 1-5), were respectively connected to the promoter P of the vector pJN44. TEF and terminator T xpr2 pJN44-RscrtB D179R , pJN44-RccarB, pJN44-XdcrtE, pJN44-PacrtE, and pJN44-SaGGPPs;

[0019] pJN44-RscrtB D179R , pJN44-RccarB and pJN44-XdcrtE, pJN44-PacrtE or pJN44-SaGGPPs were used as templates for PCR amplification of P TEF -RscrtB D179R -T xpr2 、RccarB、P TEF -XdcrtE-T xpr2 , P TEF -PacrtE-T xpr2 , P TEF -SaGGPPs-T xpr2 The promoter P was amplified by PCR using the genomic DNA of the polf strain as a template. GPD and terminator TLip1 ; P TEF -RscrtB D179R -T xpr2 , T Lip1 、RccarB、P GPD With P TEF -XdcrtE-T xpr2 , P TEF -PacrtE-T xpr2 or P TEF -SaGGPPs-T xpr2 Sequentially ligated to the ploxpura3loxp vector containing loxp and ura3 selection markers to obtain the expression vector ploxpura3loxp-RscrtB D179R -RccarB-XdcrtE, ploxpura3loxp-RscrtB D179R -RccarB-PacrtE or ploxpura3loxp-RscrtB D179R -RccarB-SaGGPPs;

[0020] (1.2) Using genomic DNA of polf strain as template, PCR amplified ERG20, IDI, ERG12, tHMGR, promoter P TEF , P GPD , P EXP1 and terminator T xpr2 、T Lip1 ; P TEF , tHMGR and T xpr2 , T Lip1 , ERG12 and P GPD , P EXP1 , IDI and T Lip1 , T xpr2 , ERG20 and P TEF , and sequentially connected to the ploxpura3loxp vector to obtain the expression vector ploxpura3loxp-tHMGR-ERG12-IDI-ERG20;

[0021] (1.3) Artificially synthesize PK and PTA genes and connect them to the promoter P of the expression plasmid pJN44. TEF and terminator T xpr2 Using synthetic plasmid as template, PCR amplified P TEF -PK-T xpr2 , PTA; PCR amplification of promoter P using genomic DNA of polf strain as template GPD and terminator T Lip1 ; P TEF -PK-T xpr2 , TLip1 ,PTA,P GPD Sequentially ligated to the ploxpleu2loxp expression plasmid containing loxp and leu2 selection markers to obtain the expression vector ploxpleu2loxp-PK-PTA;

[0022] (2) Construction of a recombinant Yarrowia lipolytica strain with high lycopene production

[0023] (2.1) Linearized ploxpura3loxp-RscrtB D179R -RccarB-XdcrtE, ploxpura3loxp-RscrtB D179R -RccarB-PacrtE or ploxpura3loxp-RscrtB D179R -RccarB-SaGGPPs were transformed into Yarrowia lipolytica polf strain to obtain the expression of RscrtB D179R , RccarB and geranylgeranyl pyrophosphate synthase genes; using the cre-loxp system to remove the ura3 selection marker in the engineering strain to obtain a recombinant Yarrowia lipolytica strain that removes the ura3 marker and expresses RscrtB D179R lipolytica engineered strain of recombinant Yarrowia lipolytica expressing , RccarB and geranylgeranyl pyrophosphate synthase genes.

[0024] In particular, the present invention also provides a recombinant Yarrowia lipolytica with high lycopene production, wherein the recombinant Yarrowia lipolytica is obtained by overexpressing the mutant RscrtB in the Yarrowia lipolytica polf strain. D179R , an engineered strain of a phytoene dehydrogenase gene RccarB from Mucor circinelloides and a geranylgeranyl pyrophosphate synthase gene XdcrtE from Phaffiarhodozyma, the recombinant Yarrowia lipolytica also expressing ERG20, IDI, ERG12 and tHMGR from Yarrowia lipolytica, the recombinant Yarrowia lipolytica also expressing a phosphoketolase gene PK from Bifidobacterium bifidum and a phosphate acetyltransferase gene PTA from Bacillus, the mutant RscrtB D179R The gene is obtained by mutating the D179 position of the phytoene synthase gene RscrtB from Mucor circinelloides from alanine to arginine.

[0025] The present invention also provides a method for constructing the above-mentioned recombinant Yarrowia lipolytica, which comprises the following steps:

[0026] (1) Expression vector construction

[0027] (1.1) Artificial synthesis of RscrtB D179R , RccarB and XdcrtE genes were connected to the promoter P of vector pJN44, respectively. TEF and terminator T xpr2 pJN44-RscrtB D179R , pJN44-RccarB and pJN44-XdcrtE;

[0028] pJN44-RscrtB D179R , pJN44-RccarB, and pJN44-XdcrtE were used as templates for PCR amplification of P TEF -RscrtB D179R -T xpr2 、RccarB、P TEF -XdcrtE-T xpr2 The promoter P was amplified by PCR using the genomic DNA of the polf strain as a template. GPD and terminator T Lip1 ; P TEF -RscrtB D179R -T xpr2 , T Lip1 、RccarB、P GPD With P TEF -XdcrtE-T xpr2 Sequentially ligated to the ploxpura3loxp vector containing loxp and ura3 selection markers to obtain the expression vector ploxpura3loxp-RscrtB D179R -RccarB-XdcrtE;

[0029] (1.2) Using genomic DNA of polf strain as template, PCR amplified ERG20, IDI, ERG12, tHMGR, promoter P TEF , P GPD , P EXP1 and terminator T xpr2 、T Lip1 ; P TEF , tHMGR and T xpr2 , T Lip1 , ERG12 and P GPD , P EXP1 , IDI and T Lip1 , T xpr2 , ERG20 and P TEF, and sequentially connected to the ploxpura3loxp vector to obtain the expression vector ploxpura3loxp-tHMGR-ERG12-IDI-ERG20;

[0030] (1.3) Artificially synthesize PK and PTA genes (as shown in SEQ ID No. 6-7) and connect them to the promoter P of the expression plasmid pJN44. TEF and terminator T xpr2 Using synthetic plasmid as template, PCR amplified P TEF -PK-T xpr2 , PTA; PCR amplification of promoter P using genomic DNA of polf strain as template GPD and terminator T Lip1 ; P TEF -PK-T xpr2 , T Lip1 ,PTA,P GPD Sequentially ligated to the ploxpleu2loxp expression plasmid containing loxp and leu2 selection markers to obtain the expression vector ploxpleu2loxp-PK-PTA;

[0031] (2) Construction of a recombinant Yarrowia lipolytica strain with high lycopene production

[0032] (2.1) Linearized ploxpura3loxp-RscrtB D179R -RccarB-XdcrtE was transformed into Yarrowia lipolytica polf strain to obtain the expression of RscrtB D179R , RccarB and XdcrtE genes; using the cre-loxp system to remove the ura3 selection marker in the engineering strain to obtain a recombinant Yarrowia lipolytica strain that removes the ura3 marker and expresses RscrtB D179R , RccarB and XdcrtE genes.

[0033] Linearized ploxpura3loxp-RscrtB D179R -RccarB-XdcrtE was again transferred into the engineered strain with ura3 marker removed to obtain an overexpression of RscrtB D179R , RccarB and XdcrtE genes; the ura3 marker in the strain was removed again to obtain a recombinant Yarrowia lipolytica strain that overexpresses RscrtB D179R , RccarB and XdcrtE genes of the recombinant Yarrowia lipolytica engineered strain;

[0034] Linearized ploxpura3loxp-RscrtBD179R -RccarB-XdcrtE was transferred into the engineered strain for the third time, and then the ura3 marker in the strain was removed to obtain the strain with the ura3 marker removed and RscrtB overexpressed for the second time. D179R , RccarB and XdcrtE genes of the recombinant Yarrowia lipolytica engineered strain;

[0035] (2.2) The linearized ploxpura3loxp-tHMGR-ERG12-IDI-ERG20 was transferred into the engineering strain obtained in step (2.1) to obtain three overexpression strains of RscrtB. D179R , RccarB and XdcrtE genes, and expressing the recombinant Yarrowia lipolytica strain of tHMGR, ERG12, IDI and ERG20 genes;

[0036] (2.3) The linearized ploxpleu2loxp-PK-PTA was transferred into the engineered strain obtained in step (2.2) to obtain the recombinant Yarrowia lipolytica strain LY23 with high lycopene production.

[0037] The present invention also provides the use of the aforementioned recombinant Yarrowia lipolytica or the recombinant Yarrowia lipolytica obtained according to the aforementioned construction method in producing lycopene, wherein the recombinant Yarrowia lipolytica is fermented in a YPD liquid culture medium at 180-220 rpm and 28-30° C. for 4-5 days.

[0038] On the other hand, the present invention also provides the use of the aforementioned recombinant Yarrowia lipolytica or the recombinant Yarrowia lipolytica obtained according to the aforementioned construction method in producing lycopene, wherein the recombinant Yarrowia lipolytica is fermented in a 5L bioreactor containing a fermentation medium at a temperature of 28-30°C, aeration of 30-50L / min, a rotation speed of 100-650rpm, oxygen 10-20%, pH of 5.5-6.0, and glucose maintained at 5g / L for 6-8d.

[0039] According to detection, the lycopene yield of the recombinant Yarrowia lipolytica obtained in the present invention in shake flask fermentation reached 1.275 g / L (156.33 mg / g DCW).

[0040] The present invention also provides the use of the recombinant Yarrowia lipolytica in producing lycopene. In a bioreactor, the recombinant Yarrowia lipolytica is fermented for 6 to 8 days in a 5L bioreactor containing 3.5L fermentation medium at a temperature of 28 to 30°C, aeration of 30 to 50L / min, a rotation speed of 100 to 650rpm, oxygen of 10 to 20%, a pH of 5.5 to 6.0, and glucose maintained at about 5g / L.

[0041] According to the test, the lycopene yield of the recombinant Yarrowia lipolytica obtained in the present invention in a 5L fermentation tank reached 11.89 g / L (157.80 mg / g DCW).

[0042] The invention expresses the lycopene synthesis pathway gene RscrtB in the Yarrowia lipolytica polf strain D179R , RccarB, XdcrtE, tHMGR, ERG12, IDI, ERG20, PK, PTA, among which the best RscrtB was selected through molecular docking D179R Mutation. The resulting recombinant Yarrowia lipolytica achieved a lycopene yield of 1.275 g / L (156.33 mg / g DCW) in shake flask fermentation and 11.89 g / L (157.80 mg / g DCW) in a 5L fermenter, significantly ahead of the existing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a schematic diagram of the synthetic pathway of lycopene produced by Yarrowia lipolytica of the present invention;

[0044] Figure 2 The shake flask fermentation results of each strain are shown;

[0045] Figure 3 The results of 5L bioreactor fermentation of LY23 strain;

[0046] Figure 4 Docking map of RscrtB protein and GGPP compound;

[0047] Figure 5 Vector plasmid ploxpura3loxp-RscrtB D179R - RccarB-XdcrtE map;

[0048] Figure 6 Vector plasmid ploxpura3loxp-tHMGR-ERG12-IDI-ERG20 map;

[0049] Figure 7 Vector plasmid ploxpleu2loxp-PK-PTA map; DETAILED DESCRIPTION

[0050] The following examples are used to explain the technical solutions of the present invention in a non-limiting manner.

[0051] In the present invention, unless otherwise specified, "%" used to describe concentrations refers to percentage by weight, and ":" refers to weight ratio.

[0052] In the present invention, the lycopene production of each strain was detected by the following method:

[0053] After the fermentation, the cells were collected and the lycopene content was detected by HPLC.

[0054] Lycopene extraction: Take 1mL of bacterial solution, centrifuge, collect the bacteria; add 500μL of dimethyl sulfoxide, incubate at 55℃ for 15 minutes; add 500μL of acetone, incubate at 45℃ for 15 minutes, shake every two minutes; centrifuge, take the supernatant, filter it with a 0.45μm filter membrane and put it in a liquid injection bottle for testing. HPLC detection method: The chromatographic column is a C18 column (Diamonsil SB-C18 column, 4.6mm×250mm); the wavelength is: 470nm; the mobile phase is (v / v): methanol: acetonitrile: isopropanol = 30:50:20; the flow rate is: 1mL / min; the column temperature is 30℃, and the elution is carried out in an isocratic elution mode.

[0055] The present invention relates to the following culture medium:

[0056] LB liquid medium: 10 g / L peptone, 10 g / L NaCl, 5 g / L yeast powder.

[0057] LB solid medium: 10 g / L peptone, 10 g / L NaCl, 5 g / L yeast powder, 20 g / L agar powder.

[0058] YPD liquid medium: 20 g / L peptone, 10 g / L yeast powder, 50 g / L glucose.

[0059] YPD solid medium: 20g / L peptone, 10g / L yeast powder, 20g / L glucose; 20g / L agar powder.

[0060] SD-leu liquid culture medium: 50g / L glucose, 1.7g / L YNB (without amino acids and ammonium sulfate), 5g / L (NH4)2SO4, 2g / L SD-leu.

[0061] SD-leu solid culture medium: 20g / L glucose, 1.7g / L YNB (without amino acids and ammonium sulfate), 5g / L (NH4)2SO4, 2g / L SD-leu, 20g / L agar powder.

[0062] SD-ura liquid culture medium: 50 g / L glucose, 1.7 g / L YNB (without amino acids and ammonium sulfate), 5 g / L (NH4)2SO4, 2 g / L SD-ura.

[0063] SD-ura solid culture medium: 20 g / L glucose, 1.7 g / L YNB (without amino acids and ammonium sulfate), 5 g / L (NH4)2SO4, 2 g / L SD-ura, 20 g / L agar powder.

[0064] Fermentation medium: 50 g / L yeast extract, 100 g / L peptone and 100 g / L glucose.

[0065] Example 1: Screening of the sources of phytoene synthase gene and phytoene dehydrogenase gene

[0066] 1. Construction of expression vector

[0067] (1) Extraction of the original strain Yarrowia lipolytica polf genome

[0068] Take 1mL of Yarrowia lipolytica polf (ATCC cell bank) bacterial liquid, centrifuge, and discard the supernatant; add 200μL 200mMLioAc, 1% SDS solution, shake and mix; 70℃ metal bath for 15min; add an equal volume (200μL) of saturated phenol: chloroform: isoamyl alcohol (25:24:1, v / v) solution, vortex to milky white, centrifuge for 10min; aspirate the aqueous phase into a new sterilized 1.5mL centrifuge tube, add an equal volume of isopropanol, mix; place at -20℃ for 1-2h, centrifuge at 12000×g for 10min to obtain DNA; add 1mL 70vol% ethanol to wash DNA (repeat 2 times), centrifuge for 4min, and discard the supernatant; turn the centrifuge tube containing DNA upside down on clean filter paper for 15min, and 50℃ metal bath for 2min to completely evaporate the ethanol; dissolve the DNA in 50μL deionized water and store at -20℃ for later use.

[0069] (2) Gene synthesis

[0070] Phytoene synthase genes RscrtB (S71770.1), PacrtB (M87280.1), SacrtB (WP_011278542.1), RccarRP were found on NCBI. E78K(AJ250827.1); phytoene dehydrogenase gene RccarB (AJ238028.1), BtcarB (AAO46894.1), PacrtI (M87280.1), with pJN44 ([1] Guokun W, XiaochaoX, Rishikesh G, et al. Exploring fatty alcohol-producing capability of Yarrowialipolytica. [J]. Biotechnology for biofuels, 2016, 9 (1)) as the vector, were sent to Nanjing GenScript Biotechnology Co., Ltd. for optimization and synthesis (the obtained phytoene dehydrogenase gene RccarB sequence is shown in SEQ ID No. 2, and the phytoene synthase gene RscrtB sequence is shown in SEQ ID No. 9), and pJN44-RscrtB, pJN44-PacrtB, pJN44-SacrtB, pJN44-RccarRP E78K ; pJN44-RccarB, pJN44-BtcarB, pJN44-PacrtI.

[0071] (3) Construction of expression plasmid

[0072] The primers shown in Table 1 were designed and sent to Shanghai Shenggong Biotechnology Co., Ltd. for synthesis. The vector ploxpura3loxp (Guokun W, Xiaochao X, Rishikesh G, et al. Exploring fatty alcohol-producing capability of Yarrowia lipolytica. [J]. Biotechnology for biofuels, 2016, 9 (1)) was digested with restriction endonuclease SpeI at 37°C for 2 h, and electrophoresed on 1% agarose gel to cut out the desired fragments, which were then recovered using a gel recovery kit (AxyGen); the plasmids pJN44-RscrtB, pJN44-PacrtB, pJN44-SacrtB, pJN44-RccarRP were synthesized. E78K template PCR amplification P TEF -RscrtB-T xpr2 , P TEF -PacrtB-T xpr2 , P TEF -SacrtB-T xpr2 , P TEF -RccarRP E78K -T xpr2gene, and 1% agarose gel electrophoresis was performed to cut out the desired fragments and recover them from the gel; the recovered ploxpura3loxp was respectively TEF -RscrtB-T xpr2 , P TEF -PacrtB-T xpr2 , P TEF -SacrtB-T xpr2 , P TEF -RccarRP E78K -T xpr2 Through seamless connection kit (Quanshijin Biotechnology Co., Ltd. -BasicSeamless Cloning and Assembly Kit) was connected at 50°C for 15 min, transformed into E. coli Trans1 T1 (Quanshijin Biotechnology Co., Ltd.), evenly spread on LB solid medium containing 100 μg / mL ampicillin, cultured at 37°C overnight, picked a single colony to LB liquid medium containing 100 μg / mL ampicillin, cultured at 37°C, 200 rpm overnight, and extracted plasmids using a plasmid extraction kit (AxyGen) to obtain plasmids ploxpura3loxp-RscrtB, ploxpura3loxp-PacrtB, ploxpura3loxp-SacrtB, ploxpura3loxp-RccarRP E78K .

[0073] Place ploxpura3loxp-RscrtB, ploxpura3loxp-PacrtB, ploxpura3loxp-SacrtB, ploxpura3loxp-RccarRP E78K The fragments were digested with restriction endonuclease MIuI, electrophoresed and recovered by the same method as above;

[0074] The synthetic plasmids pJN44-RccarB, pJN44-BtcarB, and pJN44-PacrtI obtained in step (2) were used as templates to amplify the RccarB, BtcarB, and PacrtI genes by PCR, and the genomic DNA of Yarrowia lipolytica polf was used as a template to amplify the promoter P by PCR. GPD(Carina H, IMD, RKK, et al.EasyCloneYALI:CRISPR / Cas9-Based SyntheticToolbox for Engineering of the Yeast Yarrowia lipolytica.[J].Biotechnologyjournal,2018,13(9).) and terminator T Lip1 (Carina H, IMD, RKK, et al. EasyCloneYALI: CRISPR / Cas9-Based Synthetic Toolbox for Engineering of the Yeast Yarrowialipolytica. [J]. Biotechnology journal, 2018, 13 (9).), electrophoresis, cut out the required fragments and recover them by gel; the recovered ploxpura3loxp-RscrtB, ploxpura3loxp-PacrtB, ploxpura3loxp-SacrtB, ploxpura3loxp-RccarRP E78K Respectively with T Lip1 , genes (RccarB, BtcarB, PacrtI), P GPD The same method as above was used for seamless ligation, transformation, culture and plasmid extraction to obtain the expression plasmids shown in Table 2.

[0075] Table 1 Synthesized primer sequences

[0076]

[0077] Table 2: Constructed expression plasmids

[0078]

[0079] 2. Construction of recombinant strains

[0080] The expression plasmids shown in Table 2 were digested with restriction endonuclease MIuI at 37°C for 2h, subjected to 1% agarose gel electrophoresis, the desired fragments were cut out, recovered using a gel recovery kit, and transformed into the Yarrowia lipolytica polf strain using a yeast transformation kit (Zymo Frozen-EZYeast Transformation II Kit), coated with SD-ura solid medium, and cultured at 30°C for 4 days to obtain the recombinant Yarrowia lipolytica strains shown in Table 3.

[0081] Table 3: Recombinant Yarrowia lipolytica strains and their lycopene production

[0082]

[0083] 3. Application of recombinant strains in lycopene production

[0084] Single colonies of the recombinant Yarrowia lipolytica strains shown in Table 3 were picked and inoculated into test tubes containing 2 mL of YPD liquid culture medium, and cultured at 30° C. and 200 r / min for 24 h; then, they were inoculated into shake flasks containing 10 mL of YPD liquid culture medium at an inoculum size of 2%, and cultured at 30° C. and 200 r / min for 96 h. Then, the cells were collected and the lycopene content was detected. The yield of each recombinant Yarrowia lipolytica strain is shown in Table 3. LY1 had the highest lycopene yield in the shake flask fermentation, reaching 0.061 g / L (6.77 mg / g DCW).

[0085] In addition, according to Table 3, the phytoene dehydrogenase gene RccarB from Mucor circinelloides has a higher ability to improve the production of lycopene in recombinant bacteria than BtcarB from Blakeslea trispora and PacrtI from Pantoea agglomerans. Therefore, in subsequent experiments, the phytoene dehydrogenase gene RccarB from Mucor circinelloides is preferably used.

[0086] Example 2: Screening of mutants of the phytoene synthase gene RscrtB

[0087] 1. Expression vector construction

[0088] (1) Molecular docking

[0089] The software AlphaFold2 was used to perform 3D structural modeling on the amino acid sequence of RscrtB, and the spatial 3D model of RscrtB was obtained. The compound model of the geranylgeranyl pyrophosphate GGPP substrate was obtained from NCBI. The 3D model of RscrtB was used as the receptor and the GGPP substrate was used as the ligand. RscrtB and GGPP were hydrogenated and the charge was balanced using Autodock 4.0. The grid box first covered all RscrtB structures, and the molecular docking was completed according to the docking algorithm of Lamarckian GA. The region with the highest frequency of active sites in RscrtB was found from different docking results, and the grid box was accurately determined. Based on the accurately determined RscrtB enzyme grid box, RscrtB and GGPP were docked again to determine the best model. According to the docking results, pyMOL was used to find the length and position of the hydrogen bond between the protein active site (residues within 6A) and GGPP (length less than 3.5), and the order of probability was arranged according to the lower the binding energy, the greater the probability, and the binding positions D179 and Y186 were found.

[0090] (2) Construction of saturation mutation plasmid

[0091] The partially overlapping primers shown in Table 4 were designed and sent to Shanghai Shenggong Biotechnology Co., Ltd. for synthesis. Using a site-directed mutagenesis kit (Fast Mutagenesis System, Quanshijin Biotechnology Co., Ltd.), PCR amplification was performed with the JN44-RscrtB plasmid as a template, and the amino acid bases at the D179 or Y186 position were mutated to NNK by the designed primers. After the amplification, 10 μL of PCR products were taken and detected by 1% agarose gel electrophoresis. 1 μL of DMT enzyme was added to the remaining PCR liquid to digest the methylated template in the PCR product, mixed, and incubated at 37°C for 1 hour. The plasmid was then transformed into E. coli DMT, evenly spread on LB solid medium containing 100 μg / mL ampicillin, and cultured at 37°C overnight. A single colony was picked into LB liquid medium containing 100 μg / mL ampicillin, and cultured at 37°C, 200 rpm overnight. The plasmid was extracted using a plasmid extraction kit to obtain the plasmid JN44-RscrtB in which D179 or Y186 had mutated to NNK. D179NNK , JN44-RscrtB Y186NNK .

[0092] Table 4 Primer sequences

[0093]

[0094] JN44-RscrtB D179NNK , JN44-RscrtB Y186NNKAs template, PCR amplification of P TEF -RscrtB D179NNK -T xpr2 , P TEF -RscrtB Y186NNK -T xpr2 , using pJN44-RccarB as a template, for the phytoene dehydrogenase gene RccarB from Mucor circinelloides selected in Example 1, PCR amplification of PRccarB was performed using the primers shown in Table 1, and using the genomic DNA of the polf strain as a template, PCR amplification of the promoter P GPD , terminator T Lip1 The amplified fragments were subjected to agarose gel electrophoresis, and the desired fragments were cut out and recovered from the gel; the ploxpura3loxp vector was digested with endonucleases SpeI and MIuI, and recovered from the gel; TEF -RscrtB D179NNK -T xpr2 or P TEF -RscrtB Y186NNK -T xpr2 , T Lip1 、RccarB、P GPD Sequentially seamlessly connected to the vector ploxpura3loxp to obtain the expression plasmid ploxpura3loxp-RscrtB D179NNK -RccarB and ploxpura3loxp-RscrtB Y186NNK -RccarB.

[0095] 2. Construction of recombinant strains

[0096] Plasmid ploxpura3loxp-RscrtB D179NNK -RccarB and ploxpura3loxp-RscrtB Y186NNK -RccarB were digested with restriction endonuclease MIuI at 37°C for 2h, subjected to 1% agarose gel electrophoresis, the desired fragments were cut out, recovered using a gel recovery kit, and transformed into the Yarrowia lipolytica polf strain using a yeast transformation kit, coated with SD-ura solid medium, and cultured at 30°C for 4 days to obtain the recombinant Yarrowia lipolytica strain LY13 (polf, ploxpura3loxp-RscrtB D179NNK -RccarB), LY14(polf, ploxpura3loxp-RscrtB Y186NNK -RccarB).

[0097] Different single colonies of the recombinant Yarrowia lipolytica strains LY13 and LY14 were picked and inoculated into test tubes containing 2 mL of SD-ura liquid culture medium, and cultured at 30°C and 200 r / min for 24 hours; then inoculated into a test tube containing 2 mL of SD-ura liquid culture medium at a 2% inoculum amount, cultured at 30°C and 200 r / min for 24 hours, and continuously subcultured 3-6 times to allow sufficient saturation mutation; spread on SD-ura solid culture medium, and culture at 30°C for 4 days.

[0098] 3. Application of recombinant strains in lycopene production

[0099] Pick out the darker single colonies and inoculate them into test tubes containing 2 mL of YPD liquid culture medium and mark each test tube on the plate. Culture the test tube bacteria at 30°C and 200 r / min for 24 h. Then inoculate them into a shake flask containing 10 mL of YPD liquid culture medium at a 2% inoculation rate and culture them at 30°C and 200 r / min for 96 h. Then collect the cells and detect the lycopene content.

[0100] The best single colony lycopene production of LY13 and LY14 strains is shown in Table 5.

[0101] Table 5: Lycopene production of recombinant Yarrowia lipolytica strains

[0102]

[0103] The results showed that compared with LY1, the lycopene production of the three LY13 strains was significantly improved, reaching the highest 0.125g / L (13.63mg / g DCW), while the lycopene production of the LY14 strain was not significantly improved.

[0104] It can be seen that although the mutation sites are predicted by the existing methods, the Y186 mutation of the RscrtB gene does not improve the lycopene production of the recombinant bacteria, while the D179 mutation effectively increases the lycopene production of the recombinant bacteria.

[0105] 4. Construction of RscrtB mutation vector

[0106] (1) A single colony of the LY13-3 strain with the highest lycopene content was picked from the SD-ura solid plate and placed in a centrifuge tube. 200 μL of 200 mM LioAc, 1% SDS solution was added and shaken evenly. The tube was then treated at 100°C for 15 min. The supernatant after centrifugation was used as a PCR template to amplify the RscrtB fragment, which was then sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. It was confirmed that the base GAC of the 179th amino acid alanine D of the RscrtB gene of the LY13-3 strain was mutated to the base CGC of arginine R in the recombinant strain.

[0107] (2) RscrtB as shown in SEQ ID No.1 D179R The gene was synthesized using pJN44 as a vector and sent to Nanjing GenScript Biotechnology Co., Ltd. to obtain the plasmid JN44-RscrtB D179R .

[0108] JN44-RscrtB D179R As a template, PCR amplification of P was performed using the primers shown in Table 1. TEF -RscrtB D179R -T xpr2 , using pJN44-RccarB as a template, PCR amplification of RccarB was performed using the primers shown in Table 1, and using genomic DNA of the polf strain as a template, PCR amplification of the promoter P was performed using the primers shown in Table 1 GPD , terminator T Lip1 The amplified fragments were subjected to agarose gel electrophoresis, and the desired fragments were cut out and recovered from the gel; the ploxpura3loxp vector was digested with endonucleases SpeI and MIuI, and recovered from the gel; TEF -RscrtB D179R -T xpr2 、T Lip1 、RccarB、P GPD Sequentially seamlessly connected to the vector ploxpura3loxp to obtain the expression vector ploxpura3loxp-RscrtB D179R -RccarB.

[0109] Example 3: Screening of geranylgeranyl pyrophosphate synthase gene sources

[0110] 1. Expression vector construction

[0111] Geranylgeranyl pyrophosphate synthase genes XdcrtE (DQ016502.1, from Phaffia rhodozyma), PacrtE (M87280.1, from Pantoea agglomerans), SaGGPPs (D28748.1, from Sulfolobus acidocaldarius), YlGGPPs (XP_502923.1, from Yarrowia lipolytica), and AfGPS (AF120272, from Archaeoglobus fulgidus) were found on NCBI. Using pJN44 as a vector, XdcrtE, PacrtE, SaGGPPs, and AfGPS were sent to Nanjing GenScript Biotechnology Co., Ltd. for optimization and synthesis (see SEQ ID No.3, 4, 5), pJN44-XdcrtE, pJN44-PacrtE, pJN44-SaGGPPs, pJN44-AfGPS were obtained. Primers shown in Table 6 were designed and sent to Shanghai Shenggong Biological Co., Ltd. for synthesis. Polf genomic DNA was used as a template to PCR amplify YlGGPPs, and YlGGPPs was connected between the HindIII and SmaI sites of the JN44 vector to obtain plasmid pJN44-YlGGPPs. pJN44-XdcrtE, pJN44-PacrtE, pJN44-SaGGPPs, pJN44-AfGPS, pJN44-YlGGPPs were used as templates to PCR amplify p TEF -XdcrtE-t XPR2 、p TEF -PacrtE-t XPR2 、p TEF -SaGGPPs-t XPR2 、p TEF -YlGGPPs-t XPR2 、p TEF -AfGPS-t XPR2 ploxpura3loxp-RscrtB D179R -RccarB was digested with MIuI and recovered from the gel. TEF -XdcrtE-t XPR2 、p TEF -PacrtE-t XPR2 、p TEF -SaGGPPs-t XPR2 、p TEF -YlGGPPs-t XPR2 、p TEF -AfGPS-t XPR2Seamless ligation was performed to obtain the plasmids shown in Table 7.

[0112] Table 6: Primer sequences

[0113]

[0114] Table 7: Plasmid construction

[0115]

[0116] 2. Construction of recombinant strains

[0117] The plasmids shown in Table 7 were digested with restriction endonuclease MIuI at 37°C for 2h, subjected to 1% agarose gel electrophoresis, the desired fragments were cut out, recovered using a gel recovery kit, and transformed into the Yarrowia lipolytica polf strain using a yeast transformation kit, coated with SD-ura solid culture medium, and cultured at 30°C for 4 days to obtain the recombinant Yarrowia lipolytica strains shown in Table 8.

[0118] Table 8: Recombinant Yarrowia lipolytica strains and their lycopene production

[0119]

[0120] 3. Application of recombinant strains in lycopene production

[0121] Single colonies of the recombinant Yarrowia lipolytica strains shown in Table 8 were picked and inoculated into test tubes containing 2 mL of YPD liquid culture medium, and cultured at 30° C. and 200 r / min for 24 h; then, they were inoculated into shake flasks containing 10 mL of YPD liquid culture medium at an inoculum size of 2%, and cultured at 30° C. and 200 r / min for 96 h. Then, the cells were collected and the lycopene content produced was detected, as shown in Table 8.

[0122] The results showed that in the engineered strain LY13-3 (genotype polf, ploxpura3loxp-RscrtB D179R -RccarB) based on the expression of a variety of different sources of geranylgeranyl pyrophosphate synthase genes XdcrtE, PacrtE, SaGGPPs, YlGGPPs or AfGPS can significantly increase the lycopene production of the strain, among which, the insertion of XdcrtE from Phaffiarhodozyma, PacrtE from Pantoea agglomerans and SaGGPPs from Sulfolobus acidocaldarius has a significant effect, and its lycopene production is much higher than the record of the prior art. Among them, XdcrtE has the most outstanding effect, and LY15 has the highest lycopene production in test tube fermentation, reaching 0.597g / L (68.39mg / g DCW).

[0123] Example 4: RscrtB D179R , RccarB, XdcrtE gene expression frequency screening

[0124] 1. Removal of ura3 marker from LY15 strain

[0125] The JN44-cre plasmid ([1] Guokun W, Xiaochao X, Rishikesh G, et al. Exploring fatty alcohol-producing capability of Yarrowia lipolytica. [J]. Biotechnology for biofuels, 2016, 9 (1)) was transferred into the recombinant strain LY15 using a yeast transformation kit, evenly spread on SD-leu solid culture medium, and cultured at 30°C for 3 days. A single colony was inoculated in 10 mL SD-leu liquid culture medium, cultured at 30°C, 200 rpm for 36 hours, diluted and spread on SD-leu solid culture medium, and cultured at 30°C for 3 days. A single colony was streaked on SD-leu, SD-ura, and YPD solid culture media for screening. The strain that did not grow on SD-ura culture medium and grew on SD-leu and YPD culture media was a strain without the ura3 marker. The correct strain was inoculated into 10 mL of YPD liquid medium, cultured at 30°C, 200 rpm for 24 hours, and subcultured in 10 mL of new YPD liquid medium at 2% inoculum, cultured at 30°C, 200 rpm for 12 hours. After three subcultures, it was diluted and coated on YPD solid medium and cultured at 30°C for 3 days. Single colonies were streaked and screened on YPD and SD-leu solid medium. The strain that did not grow on SD-leu solid medium and grew on YPD solid medium successfully eliminated the JN44-cre plasmid. After verification, the strain LY15D from which the ura3 marker had been successfully removed was obtained.

[0126] 2. Construction of recombinant strains

[0127] Plasmid ploxpura3loxp-RscrtB D179R -RccarB-XdcrtE was digested with restriction endonuclease MIuI at 37°C for 2h, and the desired fragment was cut out by 1% agarose gel electrophoresis, recovered by gel recovery kit, and transformed into recombinant Yarrowia lipolytica strain LY15D by the method of yeast transformation kit, coated with SD-ura solid culture medium, and cultured at 30°C for 4 days to obtain recombinant strain LY20, whose genotype is LY20 (LY15D, ploxpura3loxp-RscrtB D179R -RccarB-XdcrtE).

[0128] The same method was used to remove the ura3 marker in the LY20 strain to obtain LY20D; the linearized plasmid ploxpura3loxp-RscrtB D179R -RccarB-XdcrtE was transferred into LY20D again to obtain the recombinant strain LY21, with the genotype LY21 (LY20D, ploxpura3loxp-RscrtB D179R -RccarB-XdcrtE)

[0129] 3. Application of recombinant strains in lycopene production

[0130] Single colonies of the recombinant Yarrowia lipolytica strains LY15D, LY20, LY20D, and LY21 were picked and inoculated into test tubes containing 2 mL of YPD liquid culture medium and cultured at 30°C and 200 r / min for 24 h; then, they were inoculated into shake flasks containing 10 mL of YPD liquid culture medium at an inoculum size of 2% and fermented at 30°C and 200 r / min for 96 h. Then, the cells were collected and the lycopene production was detected, as shown in Table 9.

[0131] Table 9: Strain construction and fermentation

[0132]

[0133] The results showed that the removal of the ura3 marker in the recombinant bacteria had little effect (or had a small effect) on the ability of the recombinant bacteria to produce lycopene, while the overexpression of RscrtB in the recombinant strain D179R , RccarB, and XdcrtE genes can significantly increase the yield. D179R , RccarB, and XdcrtE genes, the yield was further increased to a lesser extent.

[0134] Example 5: Enhancing the supply of precursor compounds in the MVA pathway

[0135] 1. Construction of expression vector

[0136] (1) Target genes ERG12 (YALI0B16038g, from Yarrowia lipolytica), ERG20 (YALI0E05753g, from Yarrowia lipolytica), HMG1 (YALI0E04807g, from Yarrowia lipolytica), IDI (YALI0F04015g, from Yarrowia lipolytica), leu2 (AF260230.1, from Yarrowia lipolytica), P EXP1 (MP977616.1, derived from Yarrowia lipolytica), deleting the first 493 amino acid sequence of HMG1 can avoid its N-terminal domain-mediated self-degradation, and obtain a truncated tHMGR, as shown in SEQ ID No.8.

[0137] (2) Design the primers in Table 10 and send them to Shanghai Shenggong Biological Co., Ltd. for synthesis. Use the genomic DNA of the polf strain as a template for PCR amplification of the promoter P TEF , tHMGR, terminator T xpr2 , electrophoresis, gel recovery; the plasmid vector ploxpura3loxp was digested with restriction endonuclease SpeI, electrophoresis, gel recovery; the recovered ploxpura3loxp was combined with the promoter P TEF , tHMGR, terminator T xpr2 Use seamless ligation kit to connect, transform and culture, extract plasmid, and obtain plasmid ploxpura3loxp-tHMGR.

[0138] Table 10 Primer sequences

[0139]

[0140] The terminator T was amplified by PCR using the genomic DNA of the polf strain as a template. Lip1 、ERG12、Promoter P GPD , electrophoresis, gel recovery; the plasmid ploxpura3loxp-tHMGR was digested with restriction endonuclease MIuI, electrophoresis, gel recovery; the recovered ploxpura3loxp-tHMGR was combined with the terminator T Lip1 、ERG12、Promoter P GPD Use seamless ligation kit to connect, transform and culture, extract plasmid, and obtain plasmid ploxpura3loxp-tHMGR-ERG12.

[0141] The promoter P was amplified by PCR using the genomic DNA of the polf strain as a template. EXP1 , IDI, terminator T Lip1 , electrophoresis, gel recovery; the plasmid ploxpura3loxp-tHMGR-ERG12 was digested with restriction endonuclease MIuI, electrophoresis, gel recovery; the recovered ploxpura3loxp-tHMGR-ERG12 was ligated with promoter P EXP1 , IDI, terminator T Lip1 Use seamless ligation kit to connect, transform and culture, extract plasmid, and obtain plasmid ploxpura3loxp-tHMGR-ERG12-IDI.

[0142] The terminator T was amplified by PCR using the genomic DNA of the polf strain as a template. xpr2 、ERG20、Promoter P TEF , electrophoresis, gel recovery; the plasmid ploxpura3loxp-tHMGR-ERG12-IDI was digested with restriction endonuclease MIuI, electrophoresis, gel recovery; the recovered ploxpura3loxp-tHMGR-ERG12-IDI was ligated with terminator T xpr2 、ERG20、Promoter P TEF Use seamless ligation kit to connect, transform and culture, extract plasmid, and obtain plasmid ploxpura3loxp-tHMGR-ERG12-IDI-ERG20.

[0143] 2. Construction of recombinant strains

[0144] The ura3 marker in the LY21 strain was removed using the same method as described above to obtain LY21D; the plasmid ploxpura3loxp-tHMGR-ERG12-IDI-ERG20 was digested with the restriction endonuclease MIuI at 37°C for 2h, electrophoresed by 1% agarose gel, the desired fragment was cut out, recovered using a gel recovery kit, and transformed into the recombinant Yarrowia lipolytica strain LY21D using a yeast transformation kit, coated with SD-ura solid culture medium, and cultured at 30°C for 4 days to obtain the recombinant strain LY22, whose genotype was LY22 (LY21D, ploxpura3loxp-tHMGR-ERG12-IDI-ERG20).

[0145] 3. Application of recombinant strains in lycopene production

[0146] Single colonies of the recombinant Yarrowia lipolytica strains LY21D and LY22 were picked and inoculated into test tubes containing 2 mL of YPD liquid culture medium, and cultured at 30° C. and 200 r / min for 24 h; then, they were inoculated in a shake flask containing 10 mL of YPD liquid culture medium at a 2% inoculation rate, and fermented at 30° C. and 200 r / min for 96 h. Then, the cells were collected and the lycopene content was detected. The lycopene production of LY21D was 0.847 g / L (98.12 mg / g DCW), and the lycopene production of LY22 was 1.185 g / L (138.03 mg / g DCW).

[0147] It can be seen that expressing tHMGR, ERG12, IDI and ERG20 genes in recombinant bacteria is beneficial to further increase lycopene production.

[0148] Example 6: Introducing an exogenous pathway to enhance the supply of acetyl-CoA

[0149] 1. Construction of expression vector

[0150] Using ploxpura3loxp plasmid as template, PCR amplify other genes except ura3 marker, electrophoresis and gel recovery; using plasmid pJN44 as template, PCR amplify leu2 gene, electrophoresis and gel recovery; connecting other genes without ura3 marker recovered with leu2 gene using seamless connection kit, transformation and culture, extracting plasmid, and obtaining plasmid ploxpleu2loxp containing leu2 selection marker.

[0151] The target genes phosphoketolase gene PK (WP_003812794.1, derived from Bifidobacterium bifidum) and phosphate acetyltransferase gene PTA (WP_003243393.1, derived from Bacillus) were found on NCBI, and pJN44 was used as a vector and sent to Nanjing GenScript Biotechnology Co., Ltd. for optimization and synthesis (as shown in SEQ ID No. 6-7), obtaining pJN44-PK and pJN44-PTA.

[0152] The primers shown in Table 11 were designed and sent to Shanghai Shenggong Biological Co., Ltd. for synthesis. The synthetic plasmids pJN44-PK and pJN44-PTA were used as templates for PCR amplification of P TEF -PK-T xpr2 , PTA; PCR amplification of promoter P using genomic DNA of polf strain as template GPD and terminator T Lip1 ploxpleu2loxp was digested with endonucleases SpeI and MIuI, and the gel was recovered; P TEF -PK-T xpr2 , T Lip1,PTA,P GPD Sequentially and seamlessly ligated to the ploxpleu2loxp expression plasmid to obtain the expression vector ploxpleu2loxp-PK-PTA.

[0153] Table 11 Primer sequences

[0154]

[0155] 2. Construction of recombinant strains

[0156] The plasmid ploxpleu2loxp-PK-PTA was digested with restriction endonuclease MIuI at 37°C for 2h, electrophoresed by 1% agarose gel, the desired fragment was cut out, recovered using a gel recovery kit, and transformed into the recombinant Yarrowia lipolytica strain LY22 using a yeast transformation kit, coated with SD-leu solid culture medium, and cultured at 30°C for 4 days to obtain the recombinant strain LY23, whose genotype was LY23 (LY22, ploxpleu2loxp-PK-PTA).

[0157] 3. Application of recombinant strains in lycopene production

[0158] A single colony of the recombinant Yarrowia lipolytica strain LY23 was picked and inoculated into a test tube containing 2 mL of YPD liquid culture medium, and cultured at 30°C and 200 r / min for 24 h; then it was inoculated into a shake flask containing 10 mL of YPD liquid culture medium at an inoculum size of 2%, and fermented at 30°C and 200 r / min for 96 h. The cells were then collected and the lycopene content was detected. The lycopene production of LY23 in the shake flask fermentation reached 1.275 g / L (156.33 mg / g DCW).

[0159] It can be seen that expressing the phosphoketolase gene PK from Bifidobacterium bifidum and the phosphate acetyltransferase gene PTA from Bacillus is beneficial to improving the lycopene production of recombinant yeast.

[0160] Example 7: Application of recombinant strain LY23 in lycopene production

[0161] A single colony of the recombinant Yarrowia lipolytica strain LY23 was picked and inoculated into a shake flask containing 50mL / 250YPD liquid medium, and cultured at 30°C and 200r / min for 24h to obtain seed liquid; finally, the seed liquid was inoculated into a 5L bioreactor containing 3.5L fermentation medium at a 2% inoculation amount, and the temperature was 28-30°C, ventilation was 30-50L / min, rotation speed was 100-650rpm, oxygen was 10-20%, 5M sodium hydroxide and hydrochloric acid were added to adjust the pH to 5.5-6.0, and glucose was maintained at about 5g / L, and fermented for 6-8d. (Cells were collected every 2h to detect the lycopene content and sugar content).

[0162] The lycopene production in a 5L fermenter reached 11.89 g / L (157.80 mg / g DCW).

[0163] Control Example 1: Construction of recombinant Yarrowia lipolytica strain LY15-CR

[0164] 1. Expression vector construction

[0165] Using the pJN44-XdcrtE plasmid as a template, PCR amplified p TEF -XdcrtE-t XPR2 ploxpura3loxp-RscrtB-RccarB was digested with MIuI and recovered from the gel. TEF -XdcrtE-t XPR2 By seamless ligation, the plasmid ploxpura3loxp-RscrtB-RccarB-XdcrtE was obtained.

[0166] 2. Construction of recombinant strain LY15-CR

[0167] The plasmid ploxpura3loxp-RccarB-RscrtB-XdcrtE was digested with restriction endonuclease MIuI at 37°C for 2 hours, electrophoresed by 1% agarose gel, the desired fragment was cut out, recovered using a gel recovery kit, and transformed into the recombinant Yarrowia lipolytica strain polf using a yeast transformation kit, coated with SD-ura solid culture medium, and cultured at 30°C for 4 days to obtain the recombinant strain LY15-CR, whose genotype was LY15-CR (polf, ploxpura3loxp-RccarB-RscrtB-XdcrtE).

[0168] 3. Application of recombinant strain LY15-CR in lycopene production

[0169] A single colony of the recombinant Yarrowia lipolytica strain LY15-CR was picked and inoculated into a test tube containing 2 mL of YPD liquid culture medium, and cultured at 30°C and 200 r / min for 24 h; then it was inoculated into a shake flask containing 10 mL of YPD liquid culture medium at an inoculum size of 2%, and fermented at 30°C and 200 r / min for 96 h. The cells were then collected and the lycopene content was detected. The lycopene production of LY15-CR in the test tube fermentation reached 0.371 mg / L (38.06 mg / g DCW).

[0170] The lycopene yield of LY15 was 0.597 g / L (68.39 mg / g DCW), and the yield of LY15 was about 1.6 times that of LY15-CR.

[0171] This shows that the mutant RscrtB D179R It has a significant effect in increasing the yield of lycopene produced by fermentation of recombinant Yarrowia lipolytica.

Claims

1. Phytoene synthase gene mutant RscrtB D179R , characterized in that The mutant is derived from Mucor circinelloides ( Mucor circinelloides ) The D179 position of the phytoene synthase gene RscrtB is mutated from alanine to arginine, and the nucleic acid sequence of the mutant is shown in SEQ ID No.

1.

2. The phytoene synthase gene mutant RscrtB of claim 1 D179R The invention relates to an application of the invention in improving the yield of lycopene produced by fermentation of recombinant Yarrowia lipolytica, wherein the recombinant Yarrowia lipolytica is a Yarrowia lipolytica polf strain expressing a phytoene dehydrogenase gene.

3. Overexpression of the phytoene synthase gene mutant RscrtB according to claim 1 D179R , from Mucor circinelloides ( Mucor circinelloides Phaffia rhodotorula Phaffia rhodozyma Application of the geranylgeranyl pyrophosphate synthase gene XdcrtE in improving the yield of lycopene produced by fermentation of the recombinant Yarrowia lipolytica polf strain.

4. A recombinant Yarrowia lipolytica with high lycopene production, characterized in that The recombinant Yarrowia lipolytica is obtained by expressing the phytoene synthase gene mutant RscrtB according to claim 1 in the Yarrowia lipolytica polf strain. D179R , RccarB and geranylgeranyl pyrophosphate synthase genes, the RscrtB D179R The gene is derived from Mucor circinelloides ( Mucor circinelloides ) A mutant in which the D179 position of the phytoene synthase gene RscrtB is mutated from alanine to arginine, wherein the RccarB gene is derived from Mucor circinelloides ( Mucor circinelloides ) phytoene dehydrogenase gene, the geranylgeranyl pyrophosphate synthase is selected from the group consisting of Phaffia rhodozyma XdcrtE from Pantoea agglomerans Pantoea agglomerans Sulfolobus acidocaldarius Sulfolobus acidocaldarius SaGGPPs.

5. A recombinant Yarrowia lipolytica with high lycopene production, characterized in that The recombinant Yarrowia lipolytica is obtained by overexpressing the phytoene synthase gene mutant RscrtB according to claim 1 in the Yarrowia lipolytica polf strain. D179R , from Mucor circinelloides ( Mucor circinelloides ) and the phytoene dehydrogenase gene RccarB from Phaffia rhodozyma ( Phaffia rhodozyma The engineered strain of the geranylgeranyl pyrophosphate synthase gene XdcrtE gene, the recombinant Yarrowia lipolytica also expresses the Yarrowia lipolytica ( Yarrowia lipolytica ) ERG20, IDI, ERG12 and tHMGR, the recombinant Yarrowia lipolytica also expresses ERG20, IDI, ERG12 and tHMGR from Bifidobacterium bifidum Bifidobacterium bifidum Phosphoketolase gene PK from Bacillus Bacillus The phosphate acetyltransferase gene PTA gene, the mutant RscrtB D179R The gene is derived from Mucor circinelloides ( Mucor circinelloides ) The D179 position of the phytoene synthase gene RscrtB mutated from alanine to arginine.

6. The method for constructing the recombinant Yarrowia lipolytica according to claim 4, comprising the following steps: (1) Construction of expression vector (1.1) Artificially synthesizing the phytoene synthase gene mutant RscrtB according to claim 1 D179R , RccarB, XdcrtE, PacrtE and SaGGPPs genes were connected to the promoter P of vector pJN44, respectively. TEF and terminator T xpr2 pJN44-RscrtB D179R , pJN44-RccarB, pJN44-XdcrtE, pJN44-PacrtE, and pJN44-SaGGPPs; pJN44-RscrtB D179R , pJN44-RccarB and pJN44-XdcrtE, pJN44-PacrtE or pJN44-SaGGPPs were used as templates for PCR amplification of P TEF -RscrtB D179R -T xpr2 、RccarB、P TEF -XdcrtE-T xpr2 , P TEF -PacrtE-T xpr2 , P TEF -SaGGPPs-T xpr2 The promoter P was amplified by PCR using the genomic DNA of the polf strain as a template. GPD and terminator T Lip1 ; P TEF -RscrtB D179R -T xpr2 , T Lip1 、RccarB、P GPD With P TEF -XdcrtE-T xpr2 , P TEF -PacrtE-T xpr2 or P TEF -SaGGPPs-T xpr2 Sequentially ligated to the ploxpura3loxp vector containing loxp and ura3 selection markers to obtain the expression vector ploxpura3loxp-RscrtB D179R -RccarB-XdcrtE, ploxpura3loxp-RscrtB D179R -RccarB-PacrtE or ploxpura3loxp-RscrtB D179R -RccarB-SaGGPPs; (1.2) PCR amplification of ERG20, IDI, ERG12, tHMGR, promoter P using genomic DNA of polf strain as template TEF , P GPD , P EXP1 and terminator T xpr2 , T Lip1 ; P TEF , tHMGR and T xpr2 , T Lip1 , ERG12 and P GPD , P EXP1 , IDI and T Lip1 , T xpr2 , ERG20 and P TEF , and sequentially connected to the ploxpura3loxp vector to obtain the expression vector ploxpura3loxp-tHMGR-ERG12-IDI-ERG20; (1.3) Artificially synthesize PK and PTA genes and connect them to the promoter P of the expression plasmid pJN44. TEF and terminator T xpr2 Using synthetic plasmid as template, PCR amplified P TEF -PK-T xpr2 , PTA; PCR amplification of promoter P using genomic DNA of polf strain as template GPD and terminator T Lip1 ; P TEF -PK-T xpr2 , T Lip1 ,PTA,P GPD Sequentially ligated to the ploxpleu2loxp expression plasmid containing loxp and leu2 selection markers to obtain the expression vector ploxpleu2loxp-PK-PTA; (2) Construction of a recombinant Yarrowia lipolytica strain with high lycopene production (2.1) Linearized ploxpura3loxp-RscrtB D179R -RccarB-XdcrtE, ploxpura3loxp-RscrtB D179R -RccarB-PacrtE or ploxpura3loxp-RscrtB D179R -RccarB-SaGGPPs were transformed into Yarrowia lipolytica polf strain to obtain the expression of RscrtB D179R , RccarB and geranylgeranyl pyrophosphate synthase genes; using the cre-loxp system to remove the ura3 selection marker in the engineering strain to obtain a recombinant Yarrowia lipolytica strain that removes the ura3 marker and expresses RscrtB D179R lipolytica engineered strain of recombinant Yarrowia lipolytica expressing , RccarB and geranylgeranyl pyrophosphate synthase genes.

7. The method for constructing the recombinant Yarrowia lipolytica according to claim 4, comprising the following steps: (1) Construction of expression vector (1.1) Artificially synthesizing the phytoene synthase gene mutant RscrtB according to claim 1 D179R , RccarB and XdcrtE genes were connected to the promoter P of vector pJN44, respectively. TEF and terminator T xpr2 pJN44-RscrtB D179R , pJN44-RccarB and pJN44-XdcrtE; pJN44-RscrtB D179R , pJN44-RccarB, and pJN44-XdcrtE were used as templates for PCR amplification of P TEF -RscrtB D179R -T xpr2 、RccarB、P TEF -XdcrtE-T xpr2 The promoter P was amplified by PCR using the genomic DNA of the polf strain as a template. GPD and terminator T Lip1 ; P TEF -RscrtB D179R -T xpr2 , T Lip1 、RccarB、P GPD With P TEF -XdcrtE-T xpr2 Sequentially ligated to the ploxpura3loxp vector containing loxp and ura3 selection markers to obtain the expression vector ploxpura3loxp-RscrtB D179R -RccarB-XdcrtE; (1.2) PCR amplification of ERG20, IDI, ERG12, tHMGR, promoter P using genomic DNA of polf strain as template TEF , P GPD , P EXP1 and terminator T xpr2 , T Lip1 ; P TEF , tHMGR and T xpr2 , T Lip1 , ERG12 and P GPD , P EXP1 , IDI and T Lip1 , T xpr2 , ERG20 and P TEF , and sequentially connected to the ploxpura3loxp vector to obtain the expression vector ploxpura3loxp-tHMGR-ERG12-IDI-ERG20; (1.3) Artificially synthesize PK and PTA genes and connect them to the promoter P of the expression plasmid pJN44. TEF and terminator T xpr2 Using synthetic plasmid as template, PCR amplified P TEF -PK-T xpr2 , PTA; PCR amplification of promoter P using genomic DNA of polf strain as template GPD and terminator T Lip1 ; P TEF -PK-T xpr2 , T Lip1 ,PTA,P GPD Sequentially ligated to the ploxpleu2loxp expression plasmid containing loxp and leu2 selection markers to obtain the expression vector ploxpleu2loxp-PK-PTA; (2) Construction of a recombinant Yarrowia lipolytica strain with high lycopene production (2.1) Linearized ploxpura3loxp-RscrtB D179R -RccarB-XdcrtE was transformed into Yarrowia lipolytica polf strain to obtain the expression of RscrtB D179R , RccarB and XdcrtE genes; using the cre-loxp system to remove the ura3 selection marker in the engineering strain to obtain a recombinant Yarrowia lipolytica strain that removes the ura3 marker and expresses RscrtB D179R , RccarB and XdcrtE genes of the recombinant Yarrowia lipolytica engineered strain; Linearized ploxpura3loxp-RscrtB D179R -RccarB-XdcrtE was again transferred into the engineered strain with ura3 marker removed to obtain an overexpression of RscrtB D179R , RccarB and XdcrtE genes; the ura3 marker in the strain was removed again to obtain a recombinant Yarrowia lipolytica strain that overexpresses RscrtB D179R , RccarB and XdcrtE genes of the recombinant Yarrowia lipolytica engineered strain; Linearized ploxpura3loxp-RscrtB D179R -RccarB-XdcrtE was transferred into the engineered strain for the third time, and then the ura3 marker in the strain was removed to obtain the strain with the ura3 marker removed and RscrtB overexpressed for the second time. D179R , RccarB and XdcrtE genes of the recombinant Yarrowia lipolytica engineered strain; (2.2) The linearized ploxpura3loxp-tHMGR-ERG12-IDI-ERG20 was transferred into the engineered strain obtained in step (2.1) to obtain three overexpression strains of RscrtB. D179R , RccarB and XdcrtE genes, and expressing the recombinant Yarrowia lipolytica strain of tHMGR, ERG12, IDI and ERG20 genes; (2.3) The linearized ploxpleu2loxp-PK-PTA was transferred into the engineered strain obtained in step (2.2) to obtain the recombinant Yarrowia lipolytica strain LY23 with high lycopene production.

8. Use of the recombinant Yarrowia lipolytica according to claim 4 or 5 or the recombinant Yarrowia lipolytica obtained according to the construction method of claim 6 or 7 in producing lycopene, characterized in that The recombinant Yarrowia lipolytica is fermented in a 5L bioreactor containing a fermentation medium for 6-8 days under the conditions of a temperature of 28-30°C, aeration of 30-50L / min, a rotation speed of 100-650rpm, oxygen of 10%-20%, pH of 5.5-6.0, and glucose maintained at 5g / L.

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